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Changes in Arabidopsis thaliana gene expression in response to silver nanoparticles and silver ions Rashid Kaveh, Yue-Sheng Li, Sibia Ranjbar, Rouzbeh Tehrani, Christopher L. Brueck, and Benoit Van Aken Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es402209w • Publication Date (Web): 20 Aug 2013 Downloaded from http://pubs.acs.org on August 25, 2013
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Changes in Arabidopsis thaliana gene expression in response to silver nanoparticles and
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silver ions
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Rashid Kaveh†, Yue-Sheng Li‡, Sibia Ranjbar†, Rouzbeh Tehrani†, Christopher L. Brueck†,
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Benoit Van Aken†,*
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†
Pennsylvania 19122, United States
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Department of Civil and Environmental Engineering, Temple University, Philadelphia,
‡
Expression Microarray, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, United States
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* Corresponding author phone: 215-204-7087; fax: 215-204-4696; e-mail:
[email protected] 13 14 15
Abstract: The release of silver nanoparticles (AgNPs) in the environment has raised concerns
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about their effects on living organisms, including plants. In this study, changes in gene
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expression in Arabidopsis thaliana exposed to polyvinylpyrrolidone-coated AgNPs and silver
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ions (Ag+) were analyzed using Affymetrix expression microarrays. Exposure to 5 mg AgNPs L-
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1
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reference to non-exposed plants. Exposure to 5 mg Ag+ L-1 for 10 days resulted in up-regulation
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of 84 genes and down-regulation of 53 genes by reference to non-exposed plants. Many genes
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differentially expressed by AgNPs and Ag+ were found to be involved in plant response to
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various stresses: up-regulated genes were primarily associated with response to metals and
(20 nm) for 10 days resulted in up-regulation of 286 genes and down-regulation of 81 genes by
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oxidative stress (e.g., vacuolar cation/proton exchanger, superoxide dismutase, cytochrome P-
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450-dependent oxidase, and peroxidase), while down-regulated genes were more associated with
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response to pathogens and hormonal stimuli (e.g., auxin-regulated gene involved in organ size
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(ARGOS), ethylene signaling pathway, and systemic acquired resistance (SAR) against fungi
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and bacteria). A significant overlap was observed between genes differentially expressed in
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response to AgNPs and Ag+ (13% and 21% of total up- and down-regulated genes, respectively),
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suggesting that AgNP-induced stress originates partly from silver toxicity and partly from
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nanoparticle-specific effects. Three highly up-regulated genes in the presence of AgNPs, but not
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Ag+, belong to the thalianol biosynthetic pathway, which is thought to be involved in plant
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defense system. Results from this study provide insights into the molecular mechanisms of plant
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response to AgNPs and Ag+.
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Keywords: Silver nanoparticles, silver, Arabidopsis thaliana, gene expression, microarray
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TOC/Abstract Art
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Introduction
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Engineered nanoparticles (ENPs) are utilized in an increasing number of products, including
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textiles, electronics, pharmaceuticals, cosmetics, and water treatment reagents.1 ENPs have
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recently raised environmental concerns because they are likely to be released into the
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environment as a consequence of their widespread utilization and because they have been shown
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to exert particle-size specific toxic effects on most living organisms.2,3 Effective regulation of the
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release of ENPs in the environment has been slow to emerge, because of the relatively recent
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recognition of their environmental effects, the broad range of their application (e.g., industry,
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cosmetics, medicine), and the lack of characterization of their toxic effects.4,5 In the U.S., the
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utilization and potential release of ENPs are regulated through the Environmental Protection
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Agency (EPA) Toxic Substances Control Act (TSCA). Although EPA is currently developing a
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Significant New Use Rule (SNUR) to ensure that nanoscale materials receive appropriate
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regulatory review, it appears that there is currently no regulation specific to the utilization,
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release, and maximum acceptable levels of ENPs in the environment. There is therefore a critical
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need to collect more experimental data about the ecotoxicity of different kinds of ENPs to
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support further regulatory efforts from federal agencies. Silver nanoparticles (AgNPs) constitute
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the most widely used ENPs. Primarily because of their antimicrobial properties, AgNPs have
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been used in a wide variety of products, including textiles, bandages, deodorants, baby products,
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toothpaste, air filters, and house appliances.6,7 The ecotoxicology of AgNPs is complex because
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it may be related simultaneously to silver-specific and nanoparticle-specific biological effects.8,9
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Both AgNPs and dissolved silver have been shown to be toxic for bacteria, algae, aquatic
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organisms, plants, and humans.10,11,12 As plants constitute the basis of the terrestrial food chain,
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their exposure to AgNPs has potential implications for the agriculture and human health.10
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AgNPs released from consumer products are likely to enter wastewater. In wastewater treatment
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plants, AgNPs partition between biosolids and treated water, which can be applied on
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agricultural fields through fertilization or irrigation processes.13,14 Potential uptake of AgNPs by
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agricultural plants has therefore raised concerns about contamination of the food chain, including
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humans. As observed with other kinds of ENPs, prior studies focusing on the exposure of plants
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to AgNPs have reported both positive effects (hormeosis) and negative effects (toxicity).6,7,8,9,15
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Most toxicological studies on the effects of AgNPs have been conducted through acute toxicity
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testing (short-time exposure to high dose) although environmental effects are more adequately
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assessed by chronic toxicity testing (long-time exposure to low dose). Although chronic toxicity
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is typically difficult to observe in the laboratory, molecular studies (e.g., proteomic or
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transcriptomic methods) may provide useful information about the potential long-term effects of
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exposure to environmental contaminants.16 However, the molecular bases of the toxicity and/or
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growth promoting effects of AgNPs in higher plants have received little attention. Recently,
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changes in gene expression in plants exposed to various environmental contaminants have been
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studied using the microarray technology, providing better understanding of the molecular
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mechanisms of plant response.17-20 Following a similar strategy, the objective of this study is to
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provide new insights on the transcriptional response of the model plant, Arabidopsis thaliana,
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exposed to AgNPs and Ag+ through the use of whole-genome cDNA microarrays.
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Experimental
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Chemicals. Silver nanoparticles (silver nanopowder, 99.99%, 20 nm, CAS 7440-22-4) were
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obtained from U.S. Research Nanomaterials (Houston, TX). Silver nitrate (99.9%) was obtained
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from Sigma-Aldrich (St-Louis, MO). Polyvinylpyrrolidone (molecular weight 40,000 Dalton,
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PVP40) was obtained from Sigma-Aldrich. Phytoagar was obtained from Plant Media (Dublin,
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OH). Murashige and Skoog (MS) salt base was obtained from Carolina (Burlington, NC). Other
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chemicals were of analytical grade, solvents were of HPLC grade, and they were obtained from
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Acros Chemicals (Geel, Belgium), Fischer Scientific (Pittsburgh, PA), or Sigma-Aldrich.
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Nanoparticle characterization: For AgNP characterization, fresh particle suspensions were
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prepared in 0.5-strength Murashige and Skoog (MS) medium (at concentration of 5 to 20 mg L-1
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with an equal concentration of PVP40) and dispersed by sonication for 30 min in a water bath
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(150 W). Particle suspensions were analyzed immediately after preparation and after 24 h of
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agitation at 55 °C, 150 rpm. Particle suspensions were characterized by visible spectrometry,
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dynamic light scattering (DLS), and transmission electron microscopy (TEM). Visible spectra
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(300 to 800 nm) of particle suspensions were recorded using an Agilent 8453 spectrophotometer
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(Agilent, Santa Clara, CA). The particle size distribution and zeta potential of the suspensions
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were determined by DLS using a Zetasizer Nano (Malvern, Worcester, MA) with the following
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parameters: wavelength 632.8 nm, angle 173 °, temperature 25 °C. The particle size and
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morphology were characterized by TEM using a JEM-1400 (JEOL, Peabody, MA) with a HT
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voltage of 120 kV and a beam current of 66 mA. Samples were prepared by applying and air
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drying 4 µL of particle suspension onto a 400-mesh copper grid covered with ultrathin carbon
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film on Holey carbon support film (Ted Pella, Redding, CA).
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Plant species and culture conditions. Arabidopsis thaliana, ecotype Columbia (Col-0/Redei-
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L211497), was obtained from the Arabidopsis Biological Resource Center (Ohio State
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University, Columbus, OH). Seeds were kept on a wet filter paper at 4 ºC in the dark for 24 h.
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Seeds were then surface-sterilized by immersion successively in DI water for 1 h, in 95% ethanol
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for 5 min, and 0.6% sodium hypochloride for 5 min, and were rinsed 3 times in sterile DI water.
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Seeds were germinated under sterile conditions in 10 × 10-cm Magenta boxes (5 seeds per box)
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closed with vented lids and filled with 100 mL of semi-solid nutrient medium. The medium
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consisted of 0.5-strength MS nutrient solution supplemented with 0.3% sucrose and 0.7%
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phytoagar (pH 7.2), and it was sterilized by autoclaving (121 °C, 15 min). The boxes were
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incubated at 25 ºC under white (cool) fluorescent light (0.38 ± 0.02 W ft-2) with a 16-h light/8-h
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dark photoperiod.
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Growth inhibition experiments. The inhibitory effect of AgNPs and Ag+ was tested by cultivating
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Arabidopsis plants in the presence of increasing concentrations of the toxicants (1.0, 2.5, 5.0, 10,
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and 20 mg L-1) added to the nutrient medium. After sterilization, the medium (prepared as
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described above) was cooled to 55 ºC and supplemented with the AgNPs or Ag+ (formulated as
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aqueous stock solutions). AgNP stock solution was prepared by mixing 1.0 g AgNPs L-1 (0.1%
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w/v) and 1.0 g PVP40 L-1 (0.1% w/v) with DI water and dispersing AgNPs by sonication for 30
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min in a water bath (150 W). Ag+ stock solution was prepared by dissolving 1.575 g AgNO3 L-1
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(0.1% w/v Ag) and 1.0 g PVP40 L-1 (0.1% w/v) in DI water. Two sets of non-exposed control
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plants were grown in nutrient medium (MS) only and in nutrient medium supplemented with 20
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mg PVP40 L-1 (i.e., equivalent to the highest concentration of PVP40 applied in the experiments
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with AgNPs). After 10 days of growth, plants were removed from the medium, washed with
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water to remove the excess of medium, dried by blotting, and weighted. Twenty plants were used
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for each treatment. The significance of differences between treatments was evaluated using one-
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way ANOVA (Prim 6.0, GraphPad, La Jolla, CA) followed by Tukey's multiple comparison tests
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at 95% confidence level (p < 0.05).
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Analysis of silver in plant tissues: For the determination of silver content, plants were grown and
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exposed to AgNPs and Ag+ as described above. At harvesting time, plants were washed with
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deionized water to remove silver that was neither adsorbed nor integrated in plant tissues.1 Plants
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were then separated into roots and leaves, dried at 70 °C for 24 h, and digested in 4:1
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concentrated HNO3:30% H2O2 at 70 °C for 8 h. After dilution with deionized water, the solution
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was filtrated through 0.2 µm and analyzed using an Agilent 7500i Benchtop ICP-MS (Santa
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Clara, CA). Fifteen plants were used for each treatment. Leaf or root tissues from 5 plants were
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pooled together to obtain three biological replicates that were analyzed separately. Statistical
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analyses were performed using one-way ANOVA followed by Tukey's multiple comparison
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tests.
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Gene expression analysis using microarrays. Plants were grown as described above in the
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presence of 5 mg AgNPs or Ag+ L-1 (and, in each case, 5 mg PVP40 L-1). Control plants were
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grown in the presence of 5 mg PVP40 L-1 only. After 10 days of growth, plants were removed
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from the medium, washed, immediately soaked in 4 mL of RNA Later™ (Ambion, Foster City,
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CA), and incubated at 4 ºC for 24 h following the manufacturer's guidelines. The plants were
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then dried by blotting and stored at -80 ºC until RNA extraction. RNA was extracted from whole
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plant tissues using TRIzol® Plus RNA Purification kit with on-column PureLink® DNAase
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treatment following the manufacturer's guidelines. An additional step of tissue homogenization
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was performed after addition of the TRIzol® reagent using bead beating (1-mm glass beads,
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4,200 rpm, 40 sec). Purified RNA was kept at -80 °C. RNA was quantified by the OD260 using a
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NanoDrop™ ND-2000 spectrophotometer (Vernon Hills, IL). The quality of RNA was assessed
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by the ratios OD260/OD280 and OD260/OD230 and using an Agilent 2100 Bioanalyzer (Santa Clara,
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CA). RNA samples used for microarray analysis had OD260/OD280 ratios of 2.12 – 2.17,
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OD260/OD230 ratios of 2.16 – 2.36, and RNA integrity numbers (RIN) of 8.3 – 9.1. RNA samples
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were labeled and hybridized to the Affymetrix Arabidopsis Gene 1.0 ST Arrays according to the
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manufacturer's instructions (Affymetrix, Santa Clara, CA). For each treatment, three biological
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replicate samples were used for microarray experiments. Scanned microarray images were
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analyzed using the Affymetrix Gene Expression Console with RMA (Robust Multi-array
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Average) normalization algorithm. Further statistical analyses were performed using BRB-
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ArrayTools developed by Dr. Richard Simon and BRB-ArrayTools Development Team.21 Gene
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classification into ontology categories (GO) was performed using BLAST2GO® version 2.6.4
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(Biobam Bioinformatics, Valencia, Spain).
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Reverse-transcription real-time PCR. Quantitative analysis of gene expression was performed
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for selected genes using reverse-transcription real-time PCR (RT-qPCR). Four genes
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significantly down-regulated (fold change < 0.25), 4 genes significantly up-regulated (fold
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change > 4.0), and 4 genes with moderate expression differences (fold change between 0.4 and
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2.0) upon exposure to both AgNPs and Ag+ were selected (Supplemental Information, Table S1).
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The internal standard was the housekeeping gene, mitogen-activated protein kinase 6 (MPK6)
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(AT2G43790).20 RT-qPCRs were conducted for three biological replicates per treatment using
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the same RNA as used for the microarray experiments. RNA was reverse-transcribed into cDNA
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using SuperScript® III First-Strand Synthesis system and oligo-dT primers (Invitrogen, Foster
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City, CA). Negative controls were generated by running the reactions without reverse-
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transcriptase. Gene sequences were obtained from the National Center for Biotechnology
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Information (NCBI) and used to design gene-specific real-time primers using PrimerQuest (IDT,
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Coralville, IA). When possible (for 10 of the 12 selected genes), primers were designed with one
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of the primer sequence spanning an exon-intron boundary (primer sequences are provided as
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Supplemental Information, Table S1). Real-time PCR quantification of cDNA was performed on
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a StepOnePlus™ Real-Time PCR System using SYBR® Green PCR Master Mix (Applied
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Biosystems, Foster City, CA). The amplification efficiency for each primer set was determined
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by using log10-dilutions of cDNA according to standard protocols. CT (cycle threshold) data were
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computed by the StepOnePlus™ Software (version 2.1; Applied Biosystems). The mean relative
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levels of amplification of the target genes and standard deviations were calculated based on CT
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values and amplification efficiencies using REST 2009 (version 2.0.13; Qiagen, Foster City,
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CA).22
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MIAME compliance. This article is written in compliance with the Minimum Information About
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a Microarray Experiment (MIAME) guidelines (http://www.mged.org/miame). Microarray data
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have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) with
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the accession number E-MEXP-3950.
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Results and Discussion
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Nanoparticle characterization: Particle suspensions were characterized in fresh preparation (MS
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medium) and after 24 h of agitation at 55 °C, 150 rpm to detect potential aggregation and/or
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change in properties. The spectrum of the fresh AgNP suspension (5 mg AgNPs L-1, 5 mg
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PVP40 L-1 in 0.5-strength MS medium) showed a single peak at 405 nm, which is consistent
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with uniform-sized nanoparticles with a diameter of approx. 20 nm. No significant change of the
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spectrum was observed after agitation of the suspension for 24 h at 55 °C, 150 rpm (spectra are
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presented in Supplemental Information, Figure S1). DLS analysis of the fresh AgNP suspension
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(20 mg L-1) showed a narrow size distribution with a hydrodynamic diameter of 27.1 ± 0.3 nm,
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which shifted to 29.6 ± 0.1 nm after 24 h of agitation at 55 °C. The zeta potential of the fresh
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suspension was -38.1 ± 0.3 V, which shifted to -30.9 ± 0.6 V after 24 h of agitation at 55 °C (size
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distribution diagrams are presented in Supplemental Information, Figure S2). TEM pictures of
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AgNP suspensions showed mostly spherical particles of uniform size (Figure 1). No observable
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change was recorded after agitation at 55 °C for 24 h.
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Effect of AgNPs and Ag+ on A. thaliana growth. In order to determine the potential toxic effect
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of AgNPs and Ag+ on A. thaliana, plantlets were grown in MS medium containing increasing
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concentration of both factors, ranging from 0.0 to 20 mg L-1. Figure 2 presents the average fresh
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biomass of plants exposed for 10 days to each treatment. No significant biomass difference was
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observed between the sets of non-exposed control plants (i.e., growing in MS medium only and
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in MS medium containing 20 mg PVP40 L-1). Exposure of plants to 1.0 and 2.5 mg AgNPs L-1
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for 10 days resulted in significant increase of the biomass, although exposure to higher
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concentrations (from 5.0 to 20 mg L-1) resulted in reduction of the biomass. On the other hand,
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although no significant effect was recorded in the presence of 1.0 and 2.5 mg Ag+ L-1, the
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biomass after 10 days was significantly reduced upon exposure to 5.0 mg L-1 and above. At low
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levels (1.0 and 2.5 mg L-1), exposure to AgNPs resulted in a significantly higher plant biomass
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than exposure to Ag+. No significant difference was recorded between the two treatments at
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higher levels (5.0, 10, and 20 mg L-1).
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Previous studies focusing on the impact of AgNPs on plants have reported both positive and
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negative effects. Investigating the impact of AgNPs of different sizes (from 2 to 20 nm) in barley
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(Hordeum vulgare), flax (Linum usitatissimum), and ryegrass (Lolium perenne), El-Temsah and
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Joner6 reported a significant reduction of the germination and growth rates, which were
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dependent on the plant species and concentration of AgNPs: e.g., 20-nm particles resulted in a
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noticeable reduction of the shoot length at 10 mg L-1 in flax and ryegrass, and at 20 mg L-1 in all
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three species. Another study reported a limited effect of AgNPs (approx. 30 nm) on cucumber
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(Cucumis sativus) and lettuce (Lactuca sativa) plants.15 Germination rates were reduced by 24%
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and 5% and root elongation rates were reduced by 15% and 2% after exposure to 100 mg AgNPs
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L-1 in cucumber and lettuce, respectively. Kumari et al.7 studied the cytotoxicity and
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genotoxicity of 100-nm AgNPs on onion root tip cells (Allium cepa): based on microscopic
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observations, cells exposed to increasing concentration in AgNPs (from 25 to 100 mg L-1)
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showed a decrease of the mitotic index (MI) from 60% to 28% and the occurrence chromosomal
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aberrations. Although previous studies reported mostly toxic effects of AgNPs toward plants, a
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recent report from Wang et al.9 showed that exposure of Arabidopsis and poplar (Populus
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deltoides × nigra DN34) plants to AgNPs produced beneficial effects at low concentration: e.g.,
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exposure to 25-nm AgNPs resulted in increased biomass and evapotranspiration in poplars at the
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concentration of 1.0 mg L-1, although exposure to 10 and 100 mg L-1 resulted in decrease of
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these parameters.
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Analysis of silver content in plant tissues: After 10 days of exposure to AgNPs and Ag+, plant
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tissues were dried, acid-digested, and the silver content was determined by ICP-MS. At the same
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level of exposure, the Ag concentration was significantly higher in plants dosed with Ag+ than in
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plants dosed with AgNPs. The Ag concentration was also significantly higher in exposed root
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tissues than in exposed leave tissues. A positive relationship was observed between the exposure
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dose and Ag content in root tissues, although this relationship was less apparent in exposed leaf
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tissues. Overall, these results are consistent with previous studies described in the literature. As
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an example, Wang et al.9 recently reported a higher Ag content in A. thaliana leaves exposed to
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Ag+ (approx. 8 µg g-1) than exposed to AgNPs (approx. 2 µg g-1). As observed in our study, the
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Ag concentration detected in roots was higher than in aerial parts of the plants: 68% of total Ag+
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and 24% of total AgNPs were found in root tissues, while only 2% of total Ag+ and 1% of total
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AgNPs were found in aerial parts.
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Gene expression microarrays. The transcriptional response of plants exposed to AgNPs and Ag+
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was investigated using Affymetrix whole-transcript expression microarrays. AgNP and Ag+
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concentration of 5 mg L-1, which resulted in moderate reduction of the plant biomass, was
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chosen for microarray experiments. After filtering out the genes with low-quality signals and
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conducting univariate t-tests (p < 0.001; BRB-ArrayTools), 446 and 405 genes showed
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consistent expression levels after exposure to AgNPs and Ag+, respectively. Of these, 375 and
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141 genes were expressed at significantly different levels (change fold < 0.5 or > 2.0) by
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exposure to AgNPs and Ag+, respectively: 286 genes (78%) were up-regulated and 81 genes
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(22%) were down-regulated at significant levels by exposure to AgNPs; 84 genes (60%) were
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up-regulated and 53 genes (40%) were down-regulated at significant levels by exposure to Ag+.
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A significant overlap of differentially-expressed genes was observed upon exposure to AgNPs
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and Ag+: 15 genes were up-regulated and 29 genes were down-regulated in response to both
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AgNPs and Ag+ (representing 13% and 21% of the total up- and down-regulated genes,
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respectively). This observation suggests that some of the AgNP effects on gene expression
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originate from Ag+ released by AgNPs, which was previously observed or proposed by other
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authors.9,25 As in several other studies9,23,24, we chose to expose the plants to the same
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concentrations of AgNPs and Ag+ (expressed as mg Ag L-1). This approach was motivated by the
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lack of information about the nature of the toxic effects and cellular targets of both Ag forms.
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Soluble Ag is known to be cytotoxic and AgNP toxicity likely originates partly from the release
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of soluble Ag. Besides, nanoparticles are known to exert specific 'particulate' effects that may not
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be related to Ag+ toxicity. Dimkpa et al.25 reported that exposure of sand-grown wheat plants
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(Triticum aestivum) to both 10-nm AgNPs and soluble Ag (Ag+) (2.5 mg kg-1) resulted in
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comparable biomass reduction. Interestingly, exposure to Ag+ equivalent to soluble Ag released
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from AgNPs (63 µg kg-1) did not result in observable effect. Moreover, using TEM, the authors
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observed accumulation of AgNPs inside plant tissues, suggesting that AgNPs exert toxic effects
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that are, at least in part, unrelated to the release of soluble Ag. In order to determine the
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respective effect of AgNPs and Ag+, Stampoulis et al.8 exposed zucchini plants (Cucurbita pepo)
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to bulk (powder) Ag, AgNPs (100 nm), and Ag+ (both supernatant of AgNP suspension and
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AgNO3). The authors reported that exposure to bulk Ag at 1,000 mg L-1 did not significantly
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affect the plant biomass, while exposure to 1,000 mg L-1 AgNPs reduced the biomass by approx.
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75%. On the other hand, AgNP supernatant and Ag+ (AgNO3) at concentration as low as 1.0 mg
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L-1 reduced the biomass by approx. 25%, suggesting that about half the observed phytotoxicity
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originated from the elemental nanoparticles themselves. Few studies have been conducted on the
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transcriptional response of organisms exposed to AgNPs. Analyzing gene expression in human
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cells (HeLa) exposed to AgNPs and Ag+, Xu et al.12 reported a higher number of genes up-
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regulated (62%) than down-regulated (38%) with Ag nanoparticle exposure, which is consistent
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with our results. The authors also observed that a large number of genes were differentially
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expressed in response to both AgNPs and Ag+ (85% of up-regulated genes and 68% of down-
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regulated genes). The complete list of Arabidopsis genes up- and down-regulated by exposure to
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AgNPs and Ag+ is provided as Supplemental Information (Tables S2 and S3).
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Reverse-transcription real-time PCR. In order to validate the microarray results, quantitative
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analysis of gene expression was performed on selected genes using RT-qPCR. Figure 4 shows
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the plots of the expression levels of the selected genes as recorded using microarrays against
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their expression levels recorded using RT-qPCR. Correlations were generally satisfactory with
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Pearson's correlation coefficient of 0.96 and 0.97 for exposure to AgNPs and Ag+, respectively.
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The RT-qPCR amplification levels were corrected for the amplification efficiencies of different
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primer sets (ranging from 97.9% to 109.9%, R2 = 0.99 to 1.0) using REST 2009.
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Functional categories of differentially expressed genes. Differentially expressed genes were
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classified in gene ontology (GO) categories using the software BLAST2GO®. Distribution into
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major process and functional categories (GO level 2) showed little difference between genes up-
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and down-regulated by exposure to AgNPs and Ag+: most represented process categories for
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both up- and down-regulated genes were metabolic process, cellular process, response to
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stimulus, and biological regulation (Figure 5); most represented functional categories for both
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up- and down-regulated genes were catalytic activity, binding, nucleic acid binding transcription
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factor activity, and transporter activity (Figure 6). Two remarkable exceptions are the signaling
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process category involving only down-regulated genes (25% and 30% of total down-regulated
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genes by exposure to AgNPs and Ag+, respectively) and the electron carrier functional category
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involving (almost) only up-regulated genes (11% and 16% of total up-regulated genes by
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exposure to AgNPs and Ag+, respectively). Genes in the signaling category are mostly involved
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in hormone signaling pathways and cellular response to hormone stimuli, which may be related
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to the reduction of plant growth in response to the toxicity of AgNPs and Ag+.26 On the other
324
hand, heavy metals are known to interact with electron carriers. Up-regulation of genes in this
325
category may reflect the response of the plant to the decreased electron transport efficiency in the
326
presence of AgNPs and Ag+.27
327 328
A significant proportion of genes differentially expressed by exposure to AgNPs and/or Ag+ is
329
involved in response to stimuli (GO level 2) (49% of total up-regulated genes and 68% of total
330
down-regulated genes). Among them, most up-regulated genes (63% and 78% of genes in this
331
category up-regulated by AgNPs and Ag+, respectively) are involved in response to abiotic
332
stimuli (GO level 5), including metal ions, salts, light, starvation, oxidative stress, osmotic stress,
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and radiation. On the other hand, most down-regulated genes (74% and 62% of genes in this
334
category down-regulated by AgNPs and Ag+, respectively) are involved in response to pathogens
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and hormonal stimuli (GO level 5), including abscisic acid, auxine, cytokinin, ethylene,
336
gibberellin, jasmonic acid, and steroid hormones (Figure 6). Up-regulation of genes related to
337
abiotic stimuli likely reflects the response of the plant to AgNP/Ag+-induced stress. On the other
338
hand, down-regulation of genes related to hormones and biological stimuli can be seen as a plant
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strategy to prioritize the response to AgNP and Ag+-induced stress.26 Alternatively, hormones are
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involved primarily in the regulation of plant development and down-regulation of hormone-
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responsive genes may simply reflect attempts of the plant to limit the growth under toxic
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conditions. In addition, beside their role in plant development, hormones are known to be
343
involved in response to biological and abiotic stresses. For instance, the down-regulation of
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abscisic acid and auxin signaling pathways has been shown to play a role in various plant
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defense responses.26
346 347
Differential expression of remarkable genes. Remarkable genes discussed in this section were
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differentially expressed in response to both AgNPs and Ag+ (reflecting the effects of Ag+) or in
349
response to AgNPs only (reflecting nanoparticle-specific effects). Genes highly up-regulated
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(fold change > 4.0) in response to both AgNPs and Ag+ are involved in response to metal and
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oxidative stresses. These genes encode the following proteins: a vacuolar cation/proton
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exchanger involved in root development under metal stress (AT5G01490), a miraculin-like
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protein (MLP) (AT2G01520), two copper/zinc superoxide dismutases (AT2G28190 and
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AT1G08830), two cytochrome P-450-dependent monooxygenases (AT5G42590 and
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AT3G28740), and a peroxidase (AT3G21770). MLPs have been suggested to be involved in
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response to wounding and pathogen infection in other plant species.28 Superoxide dismutases and
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peroxidases are involved in protection against reactive oxygen species (ROS), which are
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frequently associated with metal and ENP toxicity.1 Although they are involved in many
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physiological processes, cytochrome P-450 genes have been reported to be induced by metal
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stress in Arabidopsis.29 Genes most down-regulated in response to both AgNPs and Ag+ (fold
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change ≤ 0.25) include a gene encoding an ARGOS (auxin-regulated gene involved in organ
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size) protein (AT3G59900), three genes involved in the ethylene signaling pathway
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(AT3G16770, AT5G25350, and AT2G40940), and two genes involved in systemic acquired
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resistance (SAR) against fungi (AT4G12470) and bacteria (AT5G46330). The ARGOS gene (the
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most down-regulated gene by both AgNP and Ag+, 0.08- and 0.09-fold change, respectively)
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regulates the size of lateral organs and its down-regulation likely reflects attempts of the plant to
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limit its expansion under stressed conditions.26 As suggested above, down-regulation of genes
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involved in ethylene signaling pathway and SAR can be understood as a prioritization of plant
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defense mechanisms under AgNP and Ag+-induced stresses.26 Genes differentially expressed in
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response to both AgNPs and Ag+ are likely to be implicated in response to Ag+, either directly
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added or released from AgNPs.
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On the other hand, a number of genes were differentially expressed in response to AgNPs only,
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which reflects their involvement in nanoparticle-specific responses. The most remarkable genes
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up-regulated (fold change > 4.0) specifically by AgNPs include two genes involved in salt stress
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(AT3G28220 and AT1G52000), a gene encoding a myrosinase-binding protein involved in
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defense against insects and pathogens (AT1G52040), three genes involved in the thalianol
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biosynthetic pathway (AT5G48010, AT5G48000, and AT5G47990), and a gene encoding a MLP
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involved in response to wounding (AT2G01520). The most up-regulated gene in our study (28.6-
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fold change) encodes a TRAF (tumor necrosis factor receptor-associated factor)-like protein
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involved in salt stress response. Although the relationship between salt and AgNP-induced stress
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is not readily apparent, similar functional genes were found to be induced in Arabidopsis
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exposed to other kinds of ENPs.20 The induction of genes responsive to pathogens and wounding
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may be related to mechanical damages caused by AgNPs to plant tissues.3 The three genes of the
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thalianol pathway belong to a cluster of four genes, which constitutes a rare case of gene
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clustering in higher plants.30 Although gene clusters are common in bacteria (i.e., operons), they
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are less frequent in eukaryotes and were thought until recently to be restricted to paralogs
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originating from repeated tandem genes. Only a few clusters of non-homologous, functionally-
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related genes have been detected in fungi and plants. In plants, these clusters are all involved in
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biosynthesis of stress-induced secondary metabolites that are (or are believed to be) required for
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survival under specific conditions, such as the exploitation of new environments.31
392 393
Interestingly, the most down-regulated gene by exposure to both AgNP and Ag+ in our study
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(ARGOS) was also reported down-regulated in Arabidopsis plants exposed to zinc oxide and
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fullerene nanoparticles.20 Other remarkable genes up-regulated by both AgNPs (our study) and
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zinc oxide nanoparticles20 are genes encoding a superoxide dismutase (AT1G08830) and two
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peroxidases, which are involved in response to oxidative stress (AT3G21770, AT2G18150), and
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a phytosulfokine-beta growth factor involved in response to wounding (AT3G49780).
399 400
This article presents the first whole-genome expression microarray experiment focusing on A.
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thaliana plants exposed to AgNPs and Ag+. Results from this study are believed to provide new
402
insights into the molecular mechanisms of plant response to AgNPs and Ag+.
403 404
Acknowledgments
405
This research was supported by a U.S. Department of Agriculture (USDA) - National Institute of
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Food and Agriculture (NIFA) grant (Award number: 2012-67009-19982).
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Supplemental Information
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The genes selected for the RT-qPCR validation of microarray results and the corresponding
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primer sequences are listed in Table S1. The complete list of Arabidopsis genes significantly up-
411
regulated (> 2.0) and down-regulated (< 0.5) by exposure to AgNPs and Ag+ is provided in
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Tables S2 and S3. Visible absorption spectra and DLS size-distribution diagrams of AgNP
413
suspensions are presented in Figure S1 and Figure S2, respectively. This information is available
414
free of charge via the Internet at http://pubs.acs.org/.
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Figure Captions
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Figure 1: Transmission electron micrograph of 20-nm silver nanoparticle (AgNP) suspensions
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prepared in 0.5-strength Murashige and Skoog (MS) medium with polyvinylpyrrolidone
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(molecular weight 40,000 Dalton, PVP40).
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Figure 2: Relative biomass of A. thaliana plants exposed for 10 days to various concentrations
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of silver nanoparticles (AgNPs) and silver ions (Ag+). Controls are non-exposed plants growing
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in nutrient medium (MS) only and in nutrient medium containing PVP40 (PVP). Error bars
513
represent standard deviations between 20 biological replicates. Treatments resulting in
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significantly different biomass (based on one-way ANOVA (p < 0.05) followed by Tukey's
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multiple comparison tests) are shown by different letters.
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Figure 3: Silver content measured in tissues of A. thaliana exposed to various concentrations of
518
silver nanoparticles (AgNPs) and silver ions (Ag+). Panel A: Leaves. Panel B: Roots. Controls
519
are non-exposed plants growing in nutrient medium (MS) only and in nutrient medium
520
containing PVP40 (PVP). Error bars represent standard deviations between 3 sets of pooled
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biological replicates. Treatments resulting in significantly different biomass (based on one-way
522
ANOVA (p < 0.05) followed by Tukey's multiple comparison tests) are shown by different
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letters.
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Figure 4: Evaluation of microarray expression levels using RT-qPCR. Panel A: Log2 microarray
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relative expression levels versus log2 RT-qPCR relative expression levels for exposure to silver
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nanoparticles (AgNPs). Panel B: Log2 microarray relative expression levels versus log2 RT-
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qPCR relative expression levels for exposure to silver ions (Ag+). Error bars represent the
529
standard deviations between three biological replicates.
530 531
Figure 5: Major gene ontology (GO) process categories of genes up- (fold change > 2.0) and
532
down-regulated (fold change < 0.5) in A. thaliana plants exposed for 10 days to silver
533
nanoparticles (AgNPs) and silver ions (Ag+). Distribution of genes into GO categories was
534
performed using BLAST2GO® (GO level 2). Only categories containing at least 5% of the total
535
number of genes significantly expressed in response to the treatments are shown. Panel A:
536
Genes differentially expressed by exposure to silver nanoparticles (AgNPs). Panel B: Genes
537
differentially expressed by exposure to Ag+.
538 539
Figure 6: Major gene ontology (GO) functional categories of genes up- (fold change > 2.0) and
540
down-regulated (fold change < 0.5) in A. thaliana plants exposed for 10 days to silver
541
nanoparticles (AgNPs) and silver ions (Ag+). Distribution of genes in GO categories was
542
performed using BLAST2GO® (GO level 2). Only categories containing at least 5% of the total
543
number of genes significantly expressed in response to the treatments are shown. Panel A:
544
Genes differentially expressed by exposure to silver nanoparticles (AgNPs). Panel B: Genes
545
differentially expressed by exposure to Ag+.
546
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Figure 1
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549 550 551
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on t C rol on M S t A gN rol P P 1. VP A gN 0 P mg / 2 A gN .5 L m P g A 5.0 /L gN m P g/ L 1 A gN 0 m P g 20 /L A g+ m 1 g/L A .0 m g+ 2 g/L A .5 m g+ 5. g/L A 0m g+ g /L 1 A 0m g+ g 20 /L m g/ L
C
Relative Biomass (%)
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Figure 2
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A. Leaf tissues
10 a
5 a a a a a
a
C on t C rol o M A ntro S gN lP P VP 1 A gN .0 P mg 2 / A gN .5 L P mg A 5.0 /L gN m g P 10 /L A gN m P g/ L 2 A 0m g+ 1. g/L A 0m g+ 2. g/L A 5m g+ 5. g/L 0 A g + mg 10 /L A g + mg 20 /L m g/ L
C on t C rol on M tr A S gN ol P P V 1 P A gN .0 P mg 2 / A gN .5 L P mg A 5.0 /L gN m P g/ L 1 A gN 0 m P g/ L 2 A 0m g+ g 1. /L A 0m g+ g /L 2. A 5m g+ g / 5. 0 L A g + mg 10 /L A g + mg 20 /L m g/ L
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200
20
a a
50
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Treatment
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Figure 3
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B. Root tissues
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f
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d c
b a
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0 a
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B. AG
A. SNP
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Log2(Signal) - RT-qPCR
Log2(Signal) - RT-qPCR
6 4 2 0 -2 -4
4 2 0 -2 -4 -6
-6 -5
-4
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-2
-1
0
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Figure 5
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